Xylosyltransferase and isoforms thereof

ABSTRACT

The invention relates to the isolation, purification and characterization of the enzyme xylosyltransferase (defined as “XT”). The invention describes for the first time that XT occurs in at least two isoforms (“XT-I”, “XT-II”). The invention relates furthermore to the recombinant cloning and expression of human and rat XT-I and XT-II and discloses their DNA and protein sequences. The enzymes according to the invention can be used as therapeutic agents and as diagnostic markers, e.g. for the determination of enhanced proteoglycan biosynthesis, and as biochemical markers for determination of several pathological processes such as systemic sclerosis.

The invention relates to the isolation, purification andcharacterization of the enzyme xylosyltransferase (defined as “XT”). Theinvention describes for the first time that XT occurs in at least twoisoforms (“XT-I”, “XT-II”). The invention relates furthermore to therecombinant cloning and expression of human and rat XT-I and XT-II anddiscloses their DNA and protein sequences. The enzymes according to theinvention can be used as therapeutic agents and as diagnostic markers,e.g. for the determination of enhanced proteoglycyn biosynthesis, and asbiochemical markers for determination of several pathological processessuch as systemic sclerosis.

BACKGROUND OF THE INVENTION

Proteoglycans are polyanionic molecules widely expressed in animal cellsand virtually every tissue. These abundant molecules are present in theextracellular matrix and on the cell surface and serve a wide range offunctions. They are increasingly implicated as important regulators inmany biological processes, such as extracellular matrix deposition, cellmembrane signal transfer, morphogenesis, cell migration, normal andtumor cell growth and viral infection (Ruoslahti, 1989, J. Biol. Chem.264, 13369-1372; Herold et al., 1994, J. Gen. Virol. 75, 1211-1222).Proteoglycans mediate diverse cellular processes through interactionwith a variety of protein ligands. In most of these bindingselectrostatic interactions with the glycosaminoglycan chains attached tothe core protein are involved (Kjellen & Lindahl, 1991, Annu. Rev.Biochem. 60, 443-475). Thus, the biological activity of proteoglycans isintimately related to the glycosaminoglycan biosynthesis.

The sulfated glycosaminoglycans chondroitin sulfate, heparan sulfate,heparin and dermatan sulfate are bound to the proteoglycan core proteinby a xylose-galactose-galactose binding region (Kjellen & Lindahl, 1991,l.c.). UDP-D-xylose:proteoglycan core protein β-D-

various species (Hoffmann et al., 1984, Connect. Tissue. Res. 12,151-164), and it was shown that the enzyme is secreted from theendoplasmatic reticulum into the extracellular space together withchondroitin sulfate proteoglycans (Kähnert et al., 1991, Eur. J. Clin.Chem. Clin. Biochem. 29, 624-625; Götting et al., 1999, J. Invest.Dermatol. 112, 919-924). However, the processes resulting in the releaseof XT from the endoplasmatic reticulum or the Golgi compartments and therole of XT in the extracellular matrix are not yet known.

As XT is the initial step enzyme in the biosynthesis of theglycosaminoglycan linkage region and as it is secreted into theextracellular matrix to a great extent, XT activity was proposed to be adiagnostic marker for the determination of an enhanced proteoglycanbiosynthesis and of tissue destruction (Weilke et al., 1997, Clin. Chem.43, 45-51). XT activities in the synovial fluid were found to besignificantly increased in chronic inflammatory joint diseases (Kleesieket al., 1987, J. Clin. Chem. Clin. Biochem. 25, 473-481). Recent studieshave shown that serum XT activity is a biochemical marker for thedetermination of fibrotic activity in systemic sclerosis (Götting etal.,1999, l.c.; Götting et al., 2000, Acta. Derm. Venereol. 80, 60-61.).

Up to now there was no success to isolate or characterizexylosyltransferase (XT), however some methods were described to measureand determine the activity of said enzyme from blood or body fluidsamples of patients showing pathological effects such as scleroderma orchronic joint diseases (Stoolmiller, 1972, J. Biol. Chem. 247,3525-3532). The samples were incubated with UDP-[¹⁴C]xylose and anappropiate acceptor. The incorporated radioactivity indicated the amountof XT activity. Acceptors used so far are proteoglycans, silk fibrin(Campbell et al., 1984, Anal. Biochem. 137, 505-516) and severalpeptides (Bourdon et al., 1987, Proc. Natl. Acad Sci. USA 84,3194-3198). However, the all activity tests used herein did not allowprecise determination of the lower XT activity in serum. A more specificacceptor protein is recombinant bikunin, the inhibitory component ofhuman inter-α-trypsin inhibitor. Bikunin carries a single chondroitin,which is essential for the structure of the inhibitor. The chondroitinsulfate attachement site in the N-terminal region contains all elementsresponsible for recognition by XT. The complete recognition sequence iscomposed of the amino acids a-a-a-a-G-S-G-a-b-a, with a=E or D and b=G,E or D. This sequence was confirmed by determination of theMichaelis-Menten (K_(m)) constants for in vitro xylosylation ofdifferent synthetic proteins and peptides using an enriched XTpreparation from conditioned cell culture supernatant of humanchondrocytes. The constant was determined to be 22 μM, which wasdecreased 9-fold in comparison to deglycosylated core protein frombovine cartilage (188 μM) (Brinkmann et al., 1997, J. Biol. Chem. 272,11171-11175). With recombinant bikunin as acceptor, a sensitive assaywas developed that allows precise determination of XT activity in humanserum and other body fluids (see: Weilke et al., 1997, l.c.). Using thisassay an increased xylosyltransferase activity was determined in bloodof patients with sclerotic processes of scleroderma, closely related toan elevated proteoglycan biosynthesis (Götting et al., 1999, l.c.).

The biosynthesis of glycosaminoglycans requires the coordinated actionof a large number of glycosyltransferases. Isolation and cloning ofthese glycosyltransferases has been targeted for a long time, since themajority of these enzymes are only present in minute amounts. Thestructure and sequence of the glycosyltransferases involved inbiosynthesis of the common glycosaminoglycan-protein linkage region haslong remained unknown. Recent cDNA cloning of galactosyltransferase I(Okajima et al., 1999, J. Biol. Chem. 274, 22915-22918) andglucuruonyltransferase I (Kitagawa et al., 1998, J. Biol. Chem. 273,6615-6618) identified 2 of the at least 4 enzymes involved in synthesisof the GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser structure.

Isolation, purification and characterization of XT involved inbiosynthesis of the common carbohydrate-protein linkage structure hasbeen hampered by difficulties in obtaining a sufficient amount of thesource material. Since, as above mentioned, XT can be used as anadditional biochemical marker for the determination of scleroticactivity in systemic sclerosis and some inflammatory disorders, it is areal need for an isolated, highly purified XT which can be produced byrecombinant methods, which is necessary for diagnostic and therapeuticpurposes. Moreover, the knowledge of the cDNA sequence of XT allows touse it on gene level such as in gene diagnostic or gene therapy.

Definitions

Above and below the term “XT” means xylosyltransferase(UDP-D-xylose:proteoglycan core protein β-D-xylosyltransferase (EC2.4.2.26) deriving from any origin and includes all possible isoforms,if not otherwise pointed out. The term “hXT” has the meaning of humanXT; the term “rXT” of rat XT. The terms “XT-I”, “XT-II”, “hXT-I”,“hXT-II”, “rXT-I2, “rXT-II” mean the specific isoforms 1 and 2 of XTaccording to the invention, wherein h and r have the indicated meanings.

Above and below the term “protein” means a protein, a protein fragmentor a peptide, if not otherwise explained.

Above and below the term “recombinant protein” is defined as a proteinwhich was produced by recombinant and biotechnological methods.

Above and below the term “XT” or “XT protein(s)” has the meaning of (a)protein(s) deriving from any source, if not otherwise stated out, havingthe biological activity and/or function of UDP-D-xylose:proteoglycancore protein β-D-xylosyltransferase.

Above and below the term “a protein having the biological activity ofUDP-D-xylose:proteoglyean core protein β-D-xylosyltransferase” isdefined as a protein which has XT—identical or XT—like functions andactivities and comprises XT itself and possible mutations, variants,isoforms thereof, including insertions, deletions and substitutions ofone ore more amino acids. The term includes also fragments or longerforms of XT as well as dimeric or multimeric forms thereof showing XTfunctions.

Above and below the term “isoform” means a second naturally occurringenzyme having the same biological activity as a first naturallyoccurring enzyme, however differing by another amino acid sequence.

SUMMARY OF THE INVENTION

It was found that XT can be purified 4,700-fold with 1% yield fromserum-free JAR choriocarcinoma cell culture supernatant. The isolationprocedure includes a combination of ammonium sulfate precipitation,heparin affinity chromatography, ion exchange chromatography andprotamine affinity chromatography. Amongst other proteins an unknownprotein was detected by matrix-assisted laser desorption ionization massspectrometry-time of flight analysis (MALDI-TOF) in the purified sample.The molecular weight of this isolated and purified XT protein wasdetermined as 120.000 by SDS-polyacrylamide gel electrophoresis. Theisolated protein was enzymatically cleaved by trypsin and endoproteinaseLys-C. Peptide fragments were sequenced by Edman degradation. Searcheswith the amino acid sequences in protein and EST databases showed nohomology to known sequences. XT was enriched by immunoaffunitychromatography with an immobilized antibody against a synthetic peptidededuced from the sequenced peptide fragments and was specifically elutedwith the antigen. In addition, XT was purified alternatively with anaprotinin affinity chromatography and was detected by western blotanalysis in the enzyme-containing fraction.

Based on the partial amino acid sequence of their isolated and purifiednew enzyme (XT) derived from human JAR choriocarcinoma cell culturesupernatant a novel cDNA was isolated according to the invention,encoding human XT-I enzyme using the degenerate reversetranscriptase-polymerase chain reaction method. The enzyme belongs to anovel family of glycosyltransferases having no homology to proteins ofprior art 5′- and 3′-RACE were performed to isolate a novel cDNAfragment of 3726 bp with a single open reading frame encoding at least827 amino acids with a molecular mass of 91.000. The human XT-I gene waslocated on chromosome 16p13.1 using radiation hybrid mapping, andextracts from CHO-K1 cells transfected with the XT-I cDNA in anexpression vector exhibited marked XT activity. Furthermore, a new3608-bp cDNA fragment encoding a novel protein of 865 amino acids wasalso isolated by PCR using degenerate primers based on the amino acidsequence of human XT-I. The amino acid sequence of this XT-II isoformdisplays 55% identity to the human XT-I. The XT-II gene is located onchromosome 17q21.3-17q22, and the exon/intron structure of the 15 kbgene was determined. RT-PCR analyses of XT-I and XT-II mRNA from varioustissues confirmed that both XT-I and XT-II transcripts are ubiquitouslyexpressed in the human tissues, although with different levels oftranscription. Furthermore, the cDNAs encoding XT-I and XT-II from ratwere cloned. The deduced amino acid sequences of rat xylosyltransferasesdisplayed 94% identity to the corresponding human enzyme.

Therefore, it is an object of this invention to provide the followingsubject-matters:

-   -   An isoform of UDP-D-xylose:core protein        β-D-xylosyZtransferase(XT);    -   a protein comprising a sequence of said isoform or a fragment        thereof, having the biological activity of XT;    -   a corresponding protein deriving from human or rat sources (bXT,        rXT);    -   a corresponding protein isolated from specific human tissue,        wherein said hXT has a molecular weight of 120.000 under SDS        PAGE conditions;    -   a corresponding recombinant protein, wherein said protein is        hXT-I comprising at least 827 amino acids and having the amino        acid sequence as depicted in FIG. 7B;    -   a corresponding isoform of hXT-I, termed as hXT-II, comprising        865 amino acids, exhibiting approximately 55% overall sequence        identity to human hXT-I, and having, in more detail, the amino        acid sequence as depicted in FIG. 8B.    -   a process for isolating and purifying a protein having the        biological activity of human UDP-D-xylose:proteoglycan core        protein β-D-xylosyltransferase (EC 2.4.2.26), said process is        characterized by the following steps:        -   (i) culturing from human tissue showing an enhanced XT            activity, preferably from JAR choriocarcinoma cells (ATCC            HTB-144), and harvesting the cell culture supernatant,        -   (ii) fractionated ammonium sulfate precipitation of the            supernatant of step (i),        -   (iii) heparin affinity chromatography of the precipitate of            step (ii),        -   (iv) ion exchange chromatography of the step (iii) product,            and        -   (v) affinity chromatography of the step (iv) product, and            optionally        -   (vi a SDS-Polyacrylamide gel elektrophoresis of step (v);    -   recombinant forms of hXT and rXT, the corresponding DNA        sequences (FIG. 7A, 8A, 9, 10) included as well as suitable        expression vectors and expression host cell systems;    -   antibodies directed against any of the above or below mentioned        XT proteins and their uses in immunological assays and        diagnostic tools for determining said XT proteins;    -   pharmaceutical compositions comprising a XT protein as defined        above and below, optionally together with a suitable        pharmacologically acceptable carrier, diluent or excipient;    -   uses of said XT proteins for the manufacture of a medicament for        the treatment of XT relevant diseases and disorders, wherein the        xylosyltransferase enzymes according to the invention can either        be used directly as therapeutic drug in pathological situations        where a deficiency of said enzyme and its isoforms can be        detected;    -   uses of said XT enzymes for the manufacture of a medicament for        the treatment of diseases and disorders which are caused or        accompanied by increased levels of said enzymes, e.g. in        sclerotic diseases and chronic joint diseases, wherein said        medicament is an inhibitor or antagonist of said XT proteins;    -   uses of said XI proteins as diagnostic markers for the diagnosis        of above- and below-mentioned diseases and pathological symptoms        and uses of said DNA molecules as gene markers;    -   methods of screening for compounds which are capable to inhibit        the activity of said XT proteins according to the invention        using known methods for determining the activity of XT.

DETAILED DESCRIPTION

General

UDP-D-xylose: proteoglycan core protein β-D-xylosyltransferase can beisolated and purified from JAR choriocarcinoma cell culture. Theisolated protein according to the invention is a single strandedpolypeptide with a molecular weight of 120.000. The protein wasenzymatically cleaved and eleven peptide fragments were sequenced byEdman degradation (see Examples). XT is present only in very smallamounts in animal tissues but unlike other glycosyltransferases, morethan 90% of XT is enriched in the medium of cultured cells. The highestsecretion of XT activity was measured in JAR choriocarcinoma cellculture, in which sternal cartilage chondrocytes and 21 different humancell lines were examined. To produce a highly enriched XT solution forthe isolation of XT, JAR choriocarcinoma cells were adapted to hollowfiber culture conditions using a novel bioreactor (TECNOMOUSE) andUltradoma-PF medium without serum addition as nutrient. For purificationof XT a combination of classic separation methods and new affinitymatrices was employed. Therefore, a heparin matrix was used as anaffinity ligand for the XT. When applied to immobilized heparin, XT wascompletely adsorbed at the matrix and the XT activity was eluted onlywith a high salt concentration after most contaminating proteins havebeen removed from the matrix. Protamine chloride is well-known ascationic activator for several sulfotransferases, so the effect ofprotamine chloride on the XT was investigated. An increased XT activitywas measured when protamine chloride was added to the XT assay solution(see Examples), indicating an interaction of these arginine richproteins with XT. Therefore, a protamine chloride affinity matrix wassynthesized using an aldehyde activated perfusion medium as support. Theinteraction of XT with this affinity matrix resulted in a 13.6 timesenrichment of XT. The protamine chloride affinity chromatography was themost efficient purification step during the isolation of the XT.Immobilized aprotinin, a Kunitz-type proteinase inhibitor, was found tobe another appropriate affinity matrix for enrichment of XT, as it wasable to adsorb XT quantitatively. Therefore, it was used for alternativepurification of XT. Different lines of evidence showed that the isolatedprotein corresponds to the XT: (a) XT activity was enriched usingimmobilized antibodies raised against a synthetic peptide deduced fromthe 120.000 protein, and the XT activity could be competitively elutedwith this peptide. (b) Immunoblot analysis of aprotinin affinitypurified XT corresponds with the 120.000 protein. However, non-reducingand non-denaturating gel filtration chromatography with heparinaffinity-purified XT from JAR cell culture supernatant shows anadditional peak of XT activity at a molecular weight of approximately500.000. XT is associated with proteoglycans. Treatment of XT withN-glycosidase F resulted in a decrease of the molecular weight from120.000 to 116.000, suggesting that the XT is a glycoprotein. Acomparison of the molecular mass of XT with other glycosyltransferasesinvolved in biosynthesis of proteoglycans shows that the XT is largerthan the other enzymes. Another difference is that nearly allproteoglycan glycosyltransferases are tightly bound to the membrane ofthe endoplasmic reticulum, whereas XT is secreted into the extraceuularspace. XT according to the invention contains like manyglycosyltransferases a D×D motif, suggesting that this motif is involvedin binding the metal-ion cofactor and the donator substrate (Gastinel etal., 1999, EMBO J. 18, 3546-3557). A D×D sequence was also found inpeptide 8 obtained from the enzymatically cleaved XT.

The invention discloses for the first time the molecular cloning andexpression of human and rat XT. Surprisingly, XT is found in at leasttwo isomeric forms, which are termed according to the invention as XT-Iand XT-II. Based on the amino acid sequence a novel cDNA was cloned,which encodes a protein of at least 827 amino acids with a molecularmass of 91.000. A D×D motif was identified in the XT-I amino acidsequence using hydrophobic cluster analysis. This motif has beenobserved in many glycosyltransferases, and is involved in thecoordination of a divalent cation in the binding of the nucleotide-sugar(Breton & Imberty, 1999, Curr. Opin. Struct. Biol. 9, 563-571.). Thetranslation initiation codon of the XT-I cDNA could not be yet clonedprobably due to strong secondary structures in the 5′-region of the XT-ImRNA. The XT-I protein isolated from human JAR choriocarcinoma cellculture supernatant migrated on SDS-PAGE with a molecular mass of120.000 and the protein size could be further reduced after N-glycanasedigestion. These findings indicate that the cloned cDNA represents atleast 90% of the coding region of human XT-I.

Another cDNA was completely identified from human placenta RNA, whichwas similar but not identical to the hXT-I cDNA. The new cDNA encodes aprotein of 865 amino acids with a molecular weight of 97.000. This novelprotein termed XT-I has a proline-rich region located near theamino-terminus and the type II transmembrane topology characteristic ofmany other glycosyltmnsferases cloned to date. The hXT-II protein wassimilar to the human XT-I with an overall sequence identity of 55%. Thesimilarity of the predicted amino acid sequences was low (<10%) at theamino-terminal end. However, large stretches at the C-terminal region,where the catalytic domain was found to be located inglycosyltransferases, are very conserved with an identity of more than80% in both proteins. These findings let conclude that the XT-II geneencodes another human xylosyltransferase, although the catalyticactivity and the biological role of XT-II remain to be elucidated indetail.

Since alterations in XT activity have been reported to be associatedwith fibrotic and sclerotic alterations of connective tissue (Götting etal., 1999; Götting et al., 2000, l.c.), the present findings providemolecular tools to study the function and the regulated expression ofhuman XT as well as the molecular mechanisms of these diseases.

Production of XT

JAR choriocarcinoma cells which had been adapted to growth in theserum-free Ultradoma-PF medium secreted XT activity into the cellculture supernatant. During the exponential growth XT activity and totalprotein concentration in the supernatant of a traditional cell culturesystem (T-flasks) were determined as 0.2 mU/l and 0.1 g/l, respectively.The cells were cultivated using three “Tecnomouse”® bioreactor systems.Each bioreactor contained five culture casettes. About 10⁷ cells perculture casette were inoculated and medium probes of about 0.5 ml weretaken every day to determine the viability of the cells and the glucose,and lactate concentration as well as the XT activity of the cell culturesupernatant. The cells amounted in the probes varies from 10⁵ to 10⁷cells per ml with viability between 40 and 70%. XT production increasedwithin three weeks after cell inoculation, reaching a plateau ofapproximately 30 mU/l. After three months the culture cassette wasremoved. Harvesting was carried out every two days, collecting 10 mlcell culture supernatant per culture cassette. The cell-free supernatantwas collected and stored at −75° C. Mean XT activity in the supernatantof the high density culture was determined as 29.0 mU/l, while the totalprotein concentration was estimated as 4.8 g/l. In total 18.5 l highdensity cell culture supernatant was collected and yielded 535.8 mU XT.

Isolation and Purification of XT from Cell Culture Supernatant

XT was purified form 18.5 l supernatant (equivalent to 2.000 l normalcell culture supernatant) of serum-free cultivated JAR choriocarcinomacells to an apparent homogeneity of about 4700-fold purification. Asummary of the purification steps for isolation of XT is shown inTable 1. XT activity of the crude supernatant was approximately 0.006mU/mg protein. The purification method according to the inventioncomprises four, preferably five different steps. It is possible, thatone step can be achieved more than once if necessary.

Step 1: Fractionated ammonium sulfate precipitation XT of the ammoniumsulfate precipitable fraction was dissolved in 0.46 l buffer A withsolubilization of 79.5% of the original activity.

Step 2: Heparin affinity chromatography on POROS 20 HE 4 ml ofXT-enriched solution from step 1 was loaded onto the POROS 20 HE column.XT activity was completely retained on the column. More than 70% oftotal protein passed through the column. Contaminating protein waseluted at a low NaCl concentration. 44% of the XT activity bound to theheparin matrix emerged at 0.5 M NaCl (FIG. 1A).

Step 3: Ion exchange chromatography on POROS 20 HQ 4 ml of the desaltedXT-containihg fraction from step 2 was loaded onto the POROS 20 HQcolumn equilibrated in buffer A. More than 98% of the XT activity boundto the resin. The column was then eluted stepwise with NaCl in buffer A(FIG. 1B). XT-containing fractions were collected.

Step 4: Affinity chromatography on protamine chloride The product ofstep 3 was desalted and concentrated using dia- and ultrafiltration. 100μl of the protein solution was applied to the POROS protamine chloridecolumn previously equilibrated with buffer A. Approximately 95% of thetransferase activity bound to the column, whereas 75% of thecontaminating protein did not. Additional proteins were eluted withbuffer A containing low NaCl concentrations. Enzyme activity was elutedat approximately 0.15 M NaCl (FIG. 1C). The enzyme activity was stablefor at least 6 months at −75° C.

Step 5: SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) XT-containingfractions from step 1-4 were subjected to SDS-PAGE on a 4-12% gradientpolyacrylamide gel (FIG. 2, panel A). Coomassie-stained protein bandswere excised and characterized by MALDI-TOF mass spectrometry aftertryptic digestion. The molecular weight of an unknown protein wasdetermined as 120.000 (FIG. 2, panel B). Step 5 is an optionally step.TABLE 1 Summary of single purification steps employed for isolation ofXT from 18.5 l of high density JAR cell culture supernatant. Total TotalSpec. Purifi- Recov- Volume activity protein activity cation- ery Stepml 10-3 × units mg 10-3 × units/mg fold % JAR high density 18,500 535.889,355.0 0.006 1 100 cell culture Supematant Ammonium 460 426.0 8,937.80.048 8 79 sulfate precipitation Heparin 50 108.3 473.0 0.229 40 35affinity chromatography Ion exchange 5 91.0 43.1 2.090 348 17Chromatography Protamine 1 6.83 0.24 28.458 4,743 1 affinitychromatography

Amino acid sequence analysis of XT. The MW 120.000 protein from theexcised band was digested with trypsin and endoproteinase Lys-C. Theproteolytic fragments were separated by reversed-phase HPLC, andselected peptides were subjected to automatic amino acid sequenceanalysis. Table II shows the obtained 11 amino acid sequences determinedby Edman degradation and mass spectrometry. TABLE II X represent a notidentified residue. The masses of three peptides were observed in theMALDI mass spectrum of the enzymatically digested MW 120.000 protein.Calculated mass values were obtained from the sequence obtained.observed mass calculated mass Peptide (M + H⁺) (M + H⁺) (1) E L G A K(2) E L L K (3) D M N F L K (4) I A S P P S D F G R 1045.5 1045.5 (5) LL L D (6) D F E N V D N S N F A P R 1524.7 1525.7 (7) P T F F A R (8) LQ F S E V G T D X D A (9) E L G A V K P D G R L 1152.6 1153.7 (10) E L LK R K L E Q Q E K (11) L G L L M P E KImmunochemical Detection of XT

Polyclonal antibodies against the synthetic peptide CSRQKELLKRKLEQQEKdeduced from the peptides 2 and 10 of the enzymatically cleaved MW120.000 protein were covalently bound on POROS 20 PA. About 50% of theXT activity of an applied sample was bound (FIG. 3, panel A) when apartially purified XT sample obtained by heparin affinity chromatographypurification step 2) was loaded onto the column. 58% of the adsorbed XTactivity was eluted with 150 mM NaCl, and the rest was eluted with 12 mMHCl. Furthermore, the adsorbed XT activity was also eluted from thesolid phase when 100 μl (1 mg/ml) of the synthetic peptide was added tothe mobile phase (FIG. 3, panel C). When immobilized preimmune serum wasused as affinity matrix (negative control) no XT activity was adsorbedto or eluted from the matrix (FIG. 3, panel B). The desalted XT fractionafter heparin affinity chromatography (purification step 2) was loadedon an aprotinin affinity column. The elution profile shows four majorprotein peaks (FIG. 4, panel I). 61% of the XT activity adsorbed to theaprotinin matrix emerged at 0.30 M NaCl and another 21% at 0.54 M NaCl.A single MW 120.000 band of the XT-containing fractions was detected bywestern blot analysis with the polyclonal antibodies (FIG. 4, panel B).

Determination of the Molecular Weight of XT

100 μl of heparin affinity purified XT was separated under non-reducingand non-denaturating conditions using a TSK G3000 SW column. Two XTactivity maxima were detected at MW 500.000 and 120.000 (FIG. 5, panelA). The molecular mass of the MW 120.000 protein was reduced about 3%after N-glycosidase F digestion as shown by SDS-PAGE (FIG. 5, panel B).

PCR-Based Cloning of Human XT-I cDNA

Based upon the amino acid sequence of 4 peptides degenerate primers weredesigned for cloning the XT-I cDNA (FIG. 6). When primers SPPS1 andLysc-inv were used in a PCR with the first strand cDNA of SW1353chondrosarcoma poly(A)⁺ mRNA as template, a major band of approximately690 bp was observed. After subcloning and sequencing a previouslyunknown DNA sequence was obtained from clone pCG114-29. PCRamplification with the primers DF1 and a mixture of Inv2b and Inv2cresulted in a 1724 bp fragment, which was subcloned and sequenced. Thededuced amino acid sequence of the clone pCG111-4 was identical to 6 ofthe sequenced peptides from human XT-I. The cloning strategy of rapidamplification of cDNA ends (RACE) (Chenchik et al., 1996, BioTechniques21, 526-534) was employed to clone the complete coding sequence of theXT-I cDNA. The largest DNA fragment obtained from the 3′-RACE reactionwas 1.6 kb and consisted of a 3′-untaanslated region of 1240 bp. The 5′RACE reaction was performed with different primers derived from thenucleotide sequence of XT-I cDNA, but the 5′-untranslated region ofhuman XT-I cDNA could not be cloned using this method. All DNA fragmentsobtained contained just the known cDNA sequence and an additional 80 bpof coding sequence. Thus, a PCR-based screening approach using cDNAlibraries as template was employed for cloning of the 5′-untranslatedregion. All DNA fragments obtained from the screening of 3 differenthuman cDNA libraries stopped at the same nucleotide, indicating thatstable secondary structures of the XT-I mRNA prevent the synthesis ofcDNA of the 5′ untranslated region during the reverse transcriptionreaction. However, the translation initiation codon of human XT-I couldnot be yet cloned. The combined cDNA of human XT-I contained 3726 bp(FIG. 7A) with a single open reading frame encoding at least 827 aminoacids with a molecular mass of a least 91.000. The deduced amino acidsequence contained 3 potential N-glycosylation sites (FIG. 7B). Analysisof the amino acid sequence using the hydrophobic cluster analysis(Gaboriaud et al., 1987, ) revealed the presence of a common D×D motifat position 182, which has been shown to be essential for bindingnucleotide-sugars in glycosyltransferases .

PCR-Based Cloning of Human XT-II Isoform cDNA

The degenerate primers PFF-sense and Lysc-inv1 which were designed uponthe amino acid sequence of proteolytic cleaved peptides of human XT-Iwere used in a PCR reaction with first strand cDNA of placenta poly(A)⁺mRNA as template. The PCR amplification resulted in a minor band of 1.1kb, which was cloned and sequenced. The determined nucleotide sequencewas similar but not identical to the XT-I cDNA sequence, indicating thatthe fragment encodes a XT-II isoform. To clone the complete codingsequence of the novel cDNA, the RACE strategy was employed. The cDNAfinally obtained was 3608 bp (FIG. 8A) and contained a single openreading frame encoding a protein of 865 amino acids with a molecularmass of 97.000. The 3′-untranslated region is 850 bp and a5′-untranslated region of 149 bp was identified with an in-frame stopcodon upstream of the ATG codon. A Kyte-Doolittle hydropathy analysis(Kyte & Doolittle, 1982, J. Mol. Biol. 157, 105-132) of the deducedamino acid sequence revealed one potential membrane-spanning regionconsisting of 16 hydrophobic amino acid residues at position 16 to 32,which appears to result in a type II transmembrane CHO-K1 cells andabsorbed from the medium by immunoprecipitation withanti-Xpress-antibodies and protein G agarose beads to eliminateendogenous XT activity. The enzyme-bound beads were used as an enzymesource and assayed for XT activity as shown in Table 3 (see below). Nodetectable XT activity was recovered by the affinity purification from acontrol transfection sample. The substrate specificity of therecombinant XT-I was similar to that of the XT-I isolated from humanbody fluids and cell culture supernatants. The recovered enzyme activityof the recombinantly expressed XT-I could be completely inhibited byaddition of 250 U of heparin. As specific inhibition of human XTactivity by heparin has been demonstrated previously (Kleesiek et al.,1987, l.c.), these results clearly indicate that the expressed proteinis the human XT. However, no enzymatic transfer of xylose to theacceptor peptides used in this study was observed when expressing XT-IIfused to the aminoterminal peptide tag in CHO-K1 cells.

To identify the XT-I reaction products, the bikunin peptide was labeledwith [¹⁴C]-D-xylose using the XT-I-bound beads as an enzyme source. Theproducts were subsequently subjected to the linkage-specific digestionof the bound [¹⁴C]-D-xylose with α- and β-xylosidase and alkalineβ-elimination. Incubation of the reaction products with β-xylosidaseresulted in the release of 74% of the incorporated [¹⁴C]xylose, whereasonly less than 4% of the peptide-bound xylose was digested aftertreatment with α-xylosidase. The alkaline cleavage of the O-glycosidiclinkage between the xylose and the β-hydroxyamino acid serine in thepresence of borohydride lead to the liberation of more than 97% of theenzymatically transferred [¹⁴C]xylose. In all the experiments performedno significant amount of [¹⁴C]-D-xylose was incorporated without theaddition of the bikurin peptide as acceptor. These results clearlyindicate that a xylose residue was transferred to the hydroxyamino acidserine of the bikunin peptide through a β-linkage. In conclusion theexpressed protein was identified as UDP-D-xylose:proteoglycan coreprotein β-D-xylosyltransferase (EC 2.4.2.26). TABLE 3 Xylosyltransferaseactivity of recombinant XT-I expressed in CHO-K1 cells. XT activity ofthe enzyme fractions using different acceptors and the inhibition ofenzyme activity by addition of heparin is shown. The synthetic bikunin,L-APP and L-APLP2 homologous peptides have been previously proved to begood acceptors for XT mediated xylosylation (Brinkmann et al., 1997;Götting et al., 1998). No XT activity was detected in samples frommock-transfected cells after affinity purification. n.d., not detected(detection limit, 20 μU/l). XT Activitiy Acceptor pCG227-XT +heparinRecombinant bikunin 2854 n.d. Bikunin peptide QEEGSGGGGQK 463 n.d.L-APLP2 peptide SENEGSGMAEQK 515 n.d. L-APP peptide TENEGSGLTNIK 492n.d. Peptide SGG n.d. n.d. Chondroitin sulfate A n.d. n.d. Chondroitinsulfate C n.d. n.d.Tissue-Specific Expression of XT-I and the XT-II Isoform

The expression of XT-I and XT-II isoform gene was examined in varioushuman tissues using a RT-PCR based method with normalized first-strandcDNA. Each PCR yielded a single product with predicted nucleotidelengths of 490 bp for XT-I and 717 bp for XT-II, although the amount ofthe amplified product varied (FIG. 11). Amplification of XT-I and XT-IIfragments resulted in a product visible by ethidium bromide stainingafter 36 cycles, whereas the DNA fragment corresponding to the abundantglyceraldehyde-3-phosphate dehydrogenase transcript was visible after 20cycles. The greatest abundance of XT-I expression was detected in theplacenta, kidney and pancreas and only a very weak expression wasdetected in skeletal muscle. The greatest abundance of XT-II isoform isexpressed in the kidney and pancreas.

Cellular Distribution of XT Activity in Cultured CHO-K1 Cells

After an incubation period of 3 days cultured CHO-K1 cells wereharvested and the XT activity was determined in the spent culturesupernatant and the cell lysates. 92% of the total XT activity was foundto be located in the cell culture medium (93.1 mU/10⁶ cells, SD 9.58),whereas only 2% was detected in the cell lysates (2.03 mU/10⁶ cells, SD0.44). 6% of the total XT activity (5.82 mU/10⁶ cells, SD 2.18) wasreleased from the membrane-containing fractions after addition of thedetergence Triton X-100 indicating that less than {fraction (1/10)}th ofXT is bound to cellular membranes.

Pharmaceutical and Diagnostic Use

The xylosyltransferase enzymes according to the invention can be useddirectly as therapeutic drug in pathological situations where adeficiency of said enzyme and its isoforms can be detected. As pointedout above XT enzyme may be overexpressed in some diseases and disorders.In these cases inhibitors and/or antagonists of XT-I and or XT-II can beused. Thus, the invention relates also to the use for the manufacture ofa medicament for the treatment of diseases which are caused by increasedlevels of said enzymes, wherein said medicament is an inhibitor orantagonist of xylosyltransferase.

As mentioned above the protein according to this invention can be usedas diagnostic means to evaluate pathological conditions. As used herein,the term “pharmaceutically acceptable carrier” means an inert, non toxicsolid or liquid filler, diluent or encapsulating material, not reactingadversely with the active compound or with the patient. Suitable,preferably liquid carriers, are well known in the art. The formulationsaccording to the invention may be administered as unit doses containingconventional non-toxic pharmaceutically acceptable carriers, diluents,adjuvants and vehicles which are typical for parenteral administration.

The term “parenteral” includes herein subcutaneous, intravenous,intra-articular and intratracheal injection and infusion techniques.Also other administrations such as oral administration and topicalapplication are suitable. Parenteral compositions and combinations aremost preferably adminstered intravenously either in a bolus form or as aconstant fusion according to known procedures. Tablets and capsules fororal administration contain conventional excipients such as bindingagents, fillers, diluents, tableting agents, lubricants, disintegrants,and wetting agents. The tablets may be coated according to methods wellknown in the art.

Unit doses according to the invention may contain daily required amountsof the protein according to the invention, or sub-multiples thereof tomake up the desired dose. The optimum therapeutically acceptable dosageand dose rate for a given patient (mammals, including humans) depends ona variety of factors, such as the activity of the specific activematerial employed, the age, body weight, general health, sex, diet, timeand route of administration, rate of clearance, enzyme activity(units/mg protein), the object of the treatment, i. e., therapy orprophylaxis and the nature of the disease to be treated.

Therefore, in compositions and combinations in a treated patient (invivo) a pharmaceutical effective daily dose of the protein of thisinvention (hXT, hXT-I, hXT-II) is between about 0.01 and 100 mg/kg bodyweight (based on a specific activity of 100 kU/mg), preferably between0.1 and 10 mg/kg body weight. According to the application form onesingle dose may contain between 0.5 and 10 mg of hXT.

SHORT DESCRIPTION OF THE FIGURES

FIG. 1: Purification of XT

Panel A, Heparin affinity chromatography on POROS 20 HE: The dissolvedammonium sulfate precipitate from JAR choriocarcinoma supernatant wasapplied to a POROS 20 HE column. After equilibration with buffer A, thecolumn was eluted stepwise with NaCl (------). Protein elution wasmonitored at wavelength A₂₈₀ (------), and fractions of 38 ml wereassayed for XT activity (▪). The horizontal bracket indicates fractionscollected for further purification.

Panel B Ion exchange chromatography on!POROS 20HQ: The desalted andconcentrated product from heparin affinity chromatography was loadedonto a POROS 20 HQ column. The column was washed with buffer A. Adsorbedproteins were eluted stepwise with NaCl (------). Protein elution wasmonitored at A₂₈₀ (------), and fractions of 50 ml were assayed for XTactivity (▪). The horizontal brackets indicate the fractions collectedfor further purification. Panel C, Protamine chloride affinitychromatography: The desalted and concentrated product from ion exchangechromatography was applied to a protamine chloride POROS column. After awashing step with buffer A, the column was eluted stepwise with NaCl(------). Elution was monitored at A₂₈₀ (------), and fractions of 6 mlwere assayed for XT activity (▪). The horizontal bracket indicates thefractions collected.

FIG. 2 SDS-PAGE of XT fractions at various purification steps

Panel A: XT fractions at various purification steps were subjected to4-12% gradient polyacrylamide gel for SDS-PAGE. Lane I, JAR cell culturesupernatant (crude material); lane II, ultrafiltration retentate; laneIII, dissolved ammonium sulfate precipitate; lane IV, protein elutedwith NaCl from the POROS 20 HE column; lane V, protein eluted with NaClfrom the POROS 20 HQ column; lane VI, protein eluted with NaCl from theprotamine chloride POROS column. Lane M, molecular size standard: myosin(200.000), phosphorylase b (97.000), bovine serum albumin (66.000),glutamic dehydrogenase (55.000), carbonic anhydrase (31.000), trypsininhibitor (22.000). The gel was silver-stained.

Panel B: Collected XT fractions from protamine affinity chromatographywere subjected to SDS-PAGE on a 4-12% gradient polyacrylamide gel. Thearrows indicate the stained bands corresponding to the unknown protein,which was shown to be XT (120.000) (1), hexose-6-phosphate dehydrogenase(89.000) (2), ezrin (68.600) (3), quiescin Q6 (64.000) (4), plasminogenactivator (47.000) (5), aldolase A (39.000) (6) and low molecularartefacts (7). All protein bands were excised, enzymatically digested,and the peptide mixture was characterized by MALDI-TOF massspectrometry. The gel was stained with Coomassie Brilliant Blue.

FIG. 3: Immunoaffinity chromatography of xylosyltransferase

Panel A 100 μl of the desalted XT-containing fractions eluted fromheparin affinity matrix was applied to a column of immobilizedpolyclonal antibodies. After washing with buffer D, the column waseluted as indicated by the arrow with buffer D/0.15 M NaCl (1) followedby 12 mM HCl (2). Fractions of 1 ml were collected into tubes containing1 ml of 0.1 M Tris/HCl, pH 8.0. Protein elution was monitored at 280 nm(------) and the XT activities (▪) of each fraction were assayed.

Panel B. Negative control of the immunoaffinity chromatography withimmobilized preimmune serum. For conditions see Panel A.

Panel C. Immunoaffinity chromatography with immobilized polyclonalantibodies. The column was eluted with buffer D containing 100 μl (1mg/ml) of the peptide antigen indicated by the arrow. Fractions of 1 mlwere collected into tubes containing 1 ml of 0.1 M Tris/HCl, pH 8.0.Protein elution was monitored at 280 nm (------) and the XT activities(▪) of each fraction were assayed.

FIG. 4: Aprotinin affinity chromatography of partially purified XT andimmunoblot analysis of the separated fractions

Panel A 200 μl desalted XT fraction from the heparin purification stepwas applied to an aprotinin column previously equilibrated with bufferA. After washing with buffer A, the adsorbed proteins were elutedstepwise with NaCl (------). Protein elution was monitored at A₂₈₀(------) and fractions of 2 ml were assayed for XT activity (▪).

Panel B. Aliquots of fraction 3, fraction 7, fraction 12 and fraction 16were analyzed by western blot. The MW 120.000 protein was detected inthe XT-containing fraction 7 and 12. Immunological detections wereperformed using a polyclonal rabbit antiserum raised against thesynthetic peptide CSRQKELLKRKLEQQEK deduced from the peptides 2 and 10of the enzymatically cleaved unknown protein. Prestained molecular sizestandard were myosin (190.000), BSA (64.000), glutamic dehydrogenase(51.000).

FIG. 5: Gelfiltration chromatography and N-glycosidase F digestion

Panel A. 100 μl of the XT containing fraction from heparin affinity HPLCwas applied to a TSK G3000 SW column (30 cm×7.5 mm, 10 μl particle size)previously equilibrated with buffer A/0.15 M NaCl. Elution was performedwith the same buffer. Fractions of 200 μl were collected and assayed forXT activity (▪). Protein elution was monitored at A₂₈₀ (------). Thearrows indicate the elution positions of thyroglobulin (669.000) (1),ferritin (440.000) (2), aldolase (158.000) (3), albumin (67.000) (4),ovalbumin (43.000) (5), chymotrypsinogen A (25.000) (6), andribonuclease A (13.700) (7).

Panel B. An aliquot of 1 μg of the MW 120.000 protein was analyzed bySDS-PAGE (I). Aliquots (1 μg) of the MW 120.000 protein were digestedwith 3,1×10⁻³ units of N-glycosidase F at 37° C. for 1 h (II) and 12 h(III). The samples were then subjected to SDS-PAGE, and protein bandswere detected by silver staining. The molecular size standard weremyosin (200.000), b-galactosidase (116.000), phosphorylase b (97.000).

FIG. 6: Cloning strategy for human XT-I and XT-II isoform.

(A) The peptides 4, 6, 7, and 9 are sequences that were most favorablefor the design of degenerate PCR primers. The amino acid sequence of thepeptides was obtained after proteolytic digestion of the purified humanXT. The strategy for cloning of XT-I cDNA (B) and XT-II isoform (C) isillustrated. The open reading frame of XT-I and XT-II is shown as afilled box, and the location of the peptides 4, 6, 7 and 9 isillustrated by open boxes. The location and orientation of thedegenerate primers employed for cloning of XT-I and XT-II are marked byarrows. XT-I and XT-II cDNA inserts contained within the indicatedplasmids were obtained using RT-PCR with degenerate primers (PCG111-4,pCG114-29, pCG110-7), 5′ RACE (PCG185-21, pCG212-19, pCG319-23, 3′ RACE(pCG204-38, pCG211-4) and RT-PCR with gene-specific primers (pCG176-1).H=A+C+T, Y=C+T, R=A+G, N=A+G+C+T, I=deoxyinosine.

FIG. 7A. B: Nucleotide (A) and deduced amino acid (B) sequence of thehXT-I.

The position of the peptide sequences obtained by digestion of thepurified human XT are underlined (B). Potential N-glycosylation sitesare double underlined. The D×D motif is intensified depicted. Thefigures are identical with SEQ ID 1 and 2.

FIG. 8A, B: Nucleotide (A) and amino acid (B) sequence of the hXT-IIisoform.

Potential N-glycosylation sites are double underlined (B). The putativetransmembrane domain is underlined. The figures are identical with SEQID 3 and 4.

FIG. 9: Nucleotide sequence of rXT-I (SEQ ID 5). The correspondingprotein sequence is depicted in SEQ ID 6.

FIG. 10: Nucleotide sequence of rXT-II (SEQ ID 7). The correspondingprotein sequence is depicted in SEQ ID 8.

FIG. 11: Differential expression of the XT-I, XT-II gene in humantissues. Semiquantitative RT-PCR with normalized first-strand cDNA wasused to examine the abundance of XTI-I and XT-II transcripts. A 983 bpfragment of the housekeeping gene glyceraldehyde-3-phosphatedehydrogenase cDNA was amplified as control (A). The 490 bp XT-I cDNAfragment (B) and the 717 bp fragment amplified from XT-I isoform cDNA(C) were detected in each tissue indicating that both enzymes areubiquitously expressed. The arrows indicate the expected position foreach PCR product.

The following examples describe the invention in more detail without tolimit scope of the technical teaching.

EXAMPLE 1

Materials for Isolation and Purifications. Human JAR choriocarcinomacells were purchased from ATCC (Rockville, Md.). Dried UltraDOMA-PFmedium was obtained from BioWhittaker (Vervier, Belgium) and aqua adinjecta from Braun (Melsungen, Germany). Heat-inactivated fetal calfserum, Dulbecco's phosphate-buffered saline, antibiotic/antimycoticsolution, trypsin-EDTA solution, trypan blue, protamine chloride and theBicinchoninic Acid Protein Assay Kit were purchased from Sigma(Deisenhofen, Germany). Cell culture flasks, serological pipettes, andsterile tubes were purchased from Becton Dickinson (Heidelberg,Germany). The hybrid hollow-fiber bioreactor TECNOMOUSE® was supplied byIntegra Biosciences (Fernwald, Germany), the ACA analyzer by DadeDiagnostica (Munchen, Germany) and the Super G analyzer by RLT(Mohnesee, Germany). UDP-[¹⁴C]xylose (9.88 kBq/nmol) came from DuPont(Bad Homburg, Germany), 25 mm diameter nitrocellulose discs fromSartorius (Göttingen, Germany), scintillation mixture and the liquidscintillation counter LS500TD was obtained from Beckman Coulter(Fullerton, Calif.). Ultrafiltration cells, YM1 membranes and PVDFmembranes (Immobilon P) were purchased from Millipore (Eschbom,Germany). The chromatography media POROS 20 HQ, POROS 20 HE2, POROS 20AL, POROS 20 EP and the HPLC workstation Biocad Sprint were supplied byPerseptive Biosystems (Framingham, Mass.). The gel filtration column TSKG3000 SW (30 cm×7.5 mm, 10 μm particle size) was obtained from TosoHaas(Montgomeryville, Pa.). The MALDI mass spectrometer Reflex II was fromBruker Daltonik GmbH (Bremen, Germany) and protein sequencer Procise 494cLC was purchased from PE Biosystems (Framingham, Mass.). Precastpolyacrylamide gels, buffers, and NuPAGE electrophoresis system XCell IIMini-Cell and Blot Module were from Novex (San Diego, Calif.). Thesynthetic peptide CSRQKELLKRKLEQQEK and the rabbit antiserum werepurchased from BioScience (Göttingen, Germany). Peroxidase-conjugatedaffinpure F(ab′)2 fragment goat anti-rabbit IgG (H+L) was purchased fromDianova (Hamburg, Germany). N-glycosidase F was obtained from Roche(Nannheim, Germany).

EXAMPLE 2

Cell culture. JAR choriocarcinoma cells releasing XT in the cell culturesupernatant were cultured in Ultradoma-PF medium containing 10%heat-inactivated fetal calf serum, 100 U/ml penicillin, 100 μg/mlstreptomycin and 250 ng/ml amphotericin B in a humidified atmosphere of5% CO2 and 95% air at 37° C. After incubation for 24 h in theserum-containing medium, the cell cultivation was adapted to serum-freeconditions as described previously (6). Scaling up of XT production wascarried out in three hybrid hollow-fiber bioreactors TECNOMOUSE®. Duringthe exponential growth the cells from three 175 cm 2 T-flasks(>3×10⁷cells) were detached with 0.5% trypsin and 0.2% EDTA inDulbecco's phosphate-buffered saline by incubation at 37° C. for 10 min.After centrifugation (5, min, 1000× g) of the cell suspension the cellpellet was resuspended in 10 ml 37° C. warm serum-free and protein-freeUltradoma-PF medium and washed three times with 20 ml of the samemedium. The cell suspension was drawn into a 10 ml syringe and theninoculated into the extracaprnary space (EC space) of the reactor. Thebioreactor was connected with a 2 liter medium bottle and set to 150ml/h in the recirculation mode, and the oxygenation pump was set asdescribed in the operating manual. Five days after inoculation a 10 mlsyringe was connected to the left hand EC port and 10 ml of cell culturesupernatant was harvested from the EC space. The harvesting wascontinued every two days over a period of 3 months. Glucose and lactateconcentration of the cell culture supernatant were controlled using theSuper G analyzer and the ACA analyzer, respectively. The 2 liter mediumbottle was replaced every three days. The viability of the cells wasdetermined by trypan blue exclusion.

EXAMPLE 3

Synthesis of the protamine affinity matrix. Protamine chloride wasimmobilized as ligand on POROS 20 AL. 30 mg ligand was dissolved in 10ml of 10 mM phosphate/0.15 M NaCl, pH 7.4. After the protein had beendissolved, 5 ml of 100 mM phosphate/1.50 M NaCl, pH 7.4 was added.NaCNBH₃ was dissolved in the ligand/buffer solution to a finalconcentration of 5 mg/ml and 1.0 g POROS 20 AL was suspended in the samesolution. The suspension was mixed gently on a shaker for 1 minute atRT. An additional 2 ml of 100 nM phosphate/1.50 M NaCl, pH 7.4 was addedto the suspension and the mixture was shaken continuously. This step wasrepeated every five minutes until the mixture volume was 25 ml. Afteradditional shaking for 2 h, the medium was filtered on a sintered glassfunnel. The matrix was suspended in 20 ml of 0.2 M Tris/HCl/5 g/lNaCNBH₃, pH 7.2 and mixed gently on a shaker for 30 min at RT. After themedia had been washed in a sintered glass funnel using 100 ml of 10 mMphosphate, pH 7.4, 100 ml of 1.0 MNaCl and another 100 ml of 10 mMphosphate, pH 7.4, the matrix was packed in a PEEK column (4.6×50 mm).

EXAMPLE 4

Synthesis of the aprotinin affinity matrix. Aprotinin affinity matrixwas synthesized according to the synthesis of the protamine affinitymatrix, but using aprotinin as ligand. After immobilization of theligand the matrix was packed in a PEEK column (4.6×50 mm).

EXAMPLE 5

Purification of xylosyltransferase from JAR cell culture supernatant.Fractionated ammonium sulfate precipitation and chromatography stepswere performed at RT, ultrafiltration and diafiltration were carried outat 4° C. 18.5 liters JAR cell culture supernatant collected from threehybrid hollow-fiber bioreactors, TECNOMOUSE®, each containing 5 culturecassettes, was concentrated to 800 ml with ultrafiltration cells usingYM1 cellulose membranes. The retentate was centriged at 4,000× g for 1h. The supernatant was decanted, and the pellet was discarded.

Step 1: Fractionated ammonium sulfate precipitation—Solid ammoniumsulfate was added to the supernatant to 28% saturation. After 1 h at RTthe suspension was centrifuged at 4,000× g for 2 h, the supernatant wasdecanted, and the precipitate was removed. Additional ammonium sulfatewas added to the solution to the point of 40% saturation, and thesuspension was allowed to stand for 1 h. To recover the precipitate thesupernatant was decanted after the suspension was centrifuged at 4,000×g for 2 h. Before chromatography on immobilized heparin the precipitatewas dissolved in 460 ml buffer A (20 mM sodium acetate, pH 6.0).

Step 2: Heparin affinity chromatography on POROS 20 HE2—The step 1product was passed through a 0.2 μm filter. 4.0 ml of the filtrate wasapplied to a POROS 20 HE2 column (16×100 mm) equilibrated with buffer Aat a flow rate of 40 ml/min. After washing the column with 100 ml ofbuffer A the XT activity was eluted with the same buffer containingNaCl. The NaCl concentration was increased stepwise: 20 ml buffer A/0.09M NaCl; 20 ml buffer A/0.15 M NaCl; 30 ml buffer A/0.24 M NaCl; 24 mlbuffer A/0.30 M NaCl; 24 ml buffer A/0.60 M NaCl; 24 ml buffer A/1.00 MNaCl; and 24 ml buffer A/1.89 M NaCl. Fractions of 38 ml each werecollected and the XT activity was measured. The procedure was repeated115 times by cyclic chromatography and the fractions containing XTactivity (115×38 ml) were collected.

Step 3: Ion exchange chromatography on POROS 20 HQ—Collected fractionsfrom step 2 were desalted using diafiltration with YM1 cellulosemembranes and ultrafiltration cells. After concentration of the desaltedprotein solution to 0.05 liter using analogous techniques theXT-enriched solution was subjected to ion exchange chromatography. 4.0ml of the XT solution was applied onto the POROS 20 HQ column (16×100mm) previously equilibrated with buffer A at a flow rate of 40 ml/min.The column was washed with 80 ml buffer A, and the adsorbed protein waseluted stepwise using the same buffer containing 0.07 M NaCl (88 ml),0.18 M NaCl (120 ml), and 0.36 M NaCl (120 ml) followed by a lineargradient of 0.36-1.00 M NaCl (200 ml) and another step of buffer A/2.0 MNaCl (120 ml). 50 ml fractions were collected and assayed for activityand evaluated by SDS-PAGE. Chromatography was repeated 13 times, and thefractions exhibiting XT activity (13×50 ml) were collected for affinitychromatography.

Step 4: Affinity chromatography on protanine chloride—XT-containingsolution from step 3 was desalted as described above and concentrated to5 ml by ultrafiltration with YM1 cellulose membranes. Theultrafiltration product was passed through a 0.2 μm filter. 100 μl ofthe filtrate was loaded onto a protamine chloride—POROS column (4.6×50mm) equilibrated with buffer A. The flow rate was 10 ml/min. The columnwas washed with 10.0 ml of buffer A, and the adsorbed fraction waseluted with the same buffer containing NaCl by a stepwise increase ofthe NaCl concentration: 6.6 ml buffer A/0.04 M NaCl; 6.6 ml bufferA/0,06 M NaCl; 6.6 ml buffer A/0.23 M NaCl followed by a linear gradientof 0.23-1.20 M NaCl (4.2 ml) in buffer A. Fractions of 6.0 ml werecollected, assayed for XT activity and evaluated by SDS-PAGE. Cyclicchromatography was repeated 50 times. The purified enzyme was collected,concentrated to 1.0 ml using ultrafiltration techniques and stored at−75° C.

Step 5: SDS-Polyacrylamide gel electrophoresis (SDS-PAGE)—The proteincomposition of various fractions was estimated by SDS-PAGE. Briefly,12.1 μl of sample was added to 4.7 μl of sample buffer (1.00 MTris/HCl/1.17 M sucrose, 0.28 M SDS, 2.08 mM EDTA, 0.88 mM Serva BlueG250, 0.70 mM phenol red, 0.10 M DTT, pH 8.5) and heated for 10 minutesat 99° C. After the sample had been loaded, SDS-polyacrylamide gelelectrophoresis was carried out on a 4-12% bis-tris polyacrylaride gelwith 3-(N-morpholino) propanesulfonic acid (MOPS) running buffer (1.00 MMOPS/Tris/69.3 mM SDS, 20.5 mM EDTA, pH 7.7). Protein bands weredetected by Coomassie Brilliant Blue or by silver staining. TheCoomassie bands were excised and characterized by MALDI massspectrometry and amino acid sequence analysis.

EXAMPLE 6

MALDI mass spectrometry. Coomassie-stained proteins were excised fromthe gel, repeatedly washed with H₂O and H₂O/acetonitrile and digestedovernight with trypsin and endoproteinase Lys-C at 37° C. The peptidesgenerated in the supernatant were analyzed by MALDI mass spectrometry.Sample preparation was achieved following the thin film preparationtechniques (13). Briefly, aliquots of 0.3 μl of a nitrocellulosecontaining saturated solution of a-cyano-4- hydroxycinnamic acid inacetone were deposited onto individual spots on the target.Subsequently, 0.8 μl 10% formic acid and 0.4 μl of the digest sample wasloaded on top of the thin film spots and allowed to dry slowly atambient temperature. To remove salts from the digestion buffer the spotswere washed with 10% formic acid and with H₂O. MALDI mass spectra wererecorded in the positive ion mode with delayed extraction on a Reflex IItime-of-flight instrument equipped with a SCOUT multiprobe inlet and a337 nm nitrogen laser. Ion acceleration voltage was set to 20.0 kV, thereflector voltage was set to 21.5 kV and the first extraction plate wasset to 15.4 kV. Mass spectra were obtained by averaging 50-200individual laser shots. Calibration of the spectra was performedinternally by a two-point linear fit using the autolysis products oftrypsin at m/z 842.50 and m/z 2211.10.

EXAMPLE 7

Amino acid sequence analysis of XT. The MW 120.000 Coomassie-stainedprotein was excised from the gel, repeatedly washed with H₂O andH₂O/acetonitrile and digested with trypsin and endoproteinase Lys-Covernight. For HPLC separation the excised gel fragment was extractedtwice with 0.1% TFA/60% acetonitrile. The extracted enzymatic fragmentswere separated on a capillary HPLC system equipped with a 140B solventsdelivery system (PE Biosystems), Acurate splitter (LC-Packings), UVabsorbance detector 759A (PE Biosystems), U-Z capillary flow cell(LC-Packings) and Probot fraction collector (BAI) using reversed-phasecolumn (Hypersil C18 BDS, 3 μm, 0.3×150 mm) and a linear gradient from12% acetonitrile, 0.1% TFA to 64% acetonitrile, 0.08% TFA in 90 min witha flow rate of 4 μl/min at RT. Peptide elution was monitored at 214 nmand individual fractions from the HPLC separation were analysed by MALDImass spectrometry. Sequence analysis of separated fragments wasperformed on a Procise Protein Sequencer 494 cLC using standard programssupplied by PE Biosystems.

EXAMPLE 8

Determination of XT activity. Determination of XT activity is based onthe incorporation of [14C]-D-xylose into the recombinant bikuninaccording to a previously described method (Brinkmann et al., 1997, J.Biol. Chem., 272, 11171-11175). For analysis of the substratespecificity of the recombinant xylosyltransferases synthetic peptidescontaining the XT recognition sequence were used as acceptor. Thereaction mixture for the assay contained, in a total volume of 100 μl:50 μl of XT solution, 25 mM 4-morpholineethanesulfonic acid (pH 6.5), 25mM KCl, 5 mM KF, 5 MM MgCl₂, 5 mM MnCl₂, 1.0 μM UDP-[¹⁴C]-D-xylose, and1.5 μM of the synthetic peptides. After incubation for 75 min at 37° C.,the reaction mixtures were placed on discs (25 mm diameter) ofImmobilon-AV membrane, which immobilizes even small peptides by covalentlinks (Pfund & Bourdage, 1990, Mol. Immunol. 27, 495-502), and allowedto dry. It was then washed for 10 min with 10% trichloroacetic acid andthree times with 5% trichloroacetic acid solution. Incorporatedradioactivity was determined by liquid scintillation counting.

EXAMPLE 9

Antiserum Preparation The synthetic peptide CSRQKELLKRKLEQQEK deducedfrom the sequenced peptides 2 and 10 of the enzymatically cleaved XT wassynthesized, purified by HPLC and used for immunization of rabbits(BioScience, Germany). Polyclonal antiserum was obtained by injection ofthe above antigen followed Example 10:

Preparation of solid-phase antigen. The antigen CSRQKELLKRKLEQQEK wasimmobilized on POROS 20 EP. After 1.6 mg antigen was dissolved in 1.2 mlof 10 mM phosphate/0.15 M NaCl, pH 7.4 0.60 ml of 100 mM phosphate/1.50M NaCl, pH 7.4 was added. 400 mg POROS 20 EP was suspended in thesolution and the suspension was mixed gently on a shaker at RT. At10-min intervals, five times in total, an additional 0.24 ml of 100 mMphosphate/1.50 M NaCl, pH 7.4 was added to the suspension. Afteradditional shaking for 5 days at RT the suspension was filtered on asintered glass funnel. The matrix was suspended in 4 ml 0.2 Mphosphate/0.1 M 2-mercaptoethanol, pH 7.4 and mixed on a shaker for 2 hat RT. The matrix was washed in a sintered glass funnel using 20 ml of10 mM phosphate/0.15 M NaCl, pH 7.4 and 20 ml of 1.0 M NaCl. Afteradditional washing with 20 ml of 10 mM phosphate, pH 7.4, the matrix waspacked in a PEEK column (4.6×50 mm).

EXAMPLE 11

Antibody purification Antiserum was adjusted to 50 mM Tris/HCl, pH 8.0.The solution was clarified by passage through a 0.2 μm filter. 0.4 mlfiltrate was applied at 10 ml/min to the antigen column previouslyequilibrated with buffer B (50 mM Tris/HCl, pH 8.0). After the columnwas washed with 4.1 ml buffer B and with 12.5 ml buffer B/0.15 M NaCl,the adsorbed antibody was eluted using 12.4 ml of 50 mM sodiumcitrate/0.15 M NaCl, pH 3.0 followed by 3.4 ml of 100 mM sodiumcitrate/1.5 M NaCl, pH 3.0. The eluate was collected as 10 ml fractionsin tubes containing 2 ml of 0.5 M Tris/HCl, pH 8.0, to immediatelyneutralize the citric acid.

EXAMPLE 12

Preparation of immunoaffinity column. Purified antibody was concentratedto a protein concentration of 0.3 mg/ml using ultrafiltration with YM1cellulose membranes. The antibody solution was adjusted to 10 mMphosphate/0.15 M NaCl, pH 7.4. After filtration of the solution througha 0.2 μm filter 100 μl filtrate was applied at 0.2 ml/min to a POROS 20PA column (2.1×30 mm). This step was repeated 17 times. Adsorbedantibody was cross-linked using cross-linking solution (100 mMtriethanolamine, pH 8.5). After the column was washed with 5 ml ofbuffer C (10 mM phosphate/0.15 M NaCl, pH 7.4) 2 ml cross-linkingsolution was applied at 0.5 ml/min onto the cartridge. The procedure wasrepeated 6 more times, using a total volume of 14 ml cross-linkingsolution. To block unreacted functional groups on the cross-linkingreagents 2 ml of 100 mM monoethanolamine, pH 9.0 (quenching solution),was loaded onto the cartridge at 0.5 ml/min. The column was washed using2 ml of buffer C and the crossinking step was repeated using another 2ml quenching solution. The immunoaffinity column was cycled betweenbuffer C and 12 mM HCl/0.15 M NaCl 3 times using a total volume of 12 mlof solution.

EXAMPLE 13

Immunoaffinity column purification of XT. XT-containing fractions elutedfrom the heparin affinity matrix were desalted using diafiltration withYM1 cellulose membranes and passed through a 0.2 μm filter. 100 μl ofthis XT-sample was applied to the immunoaffinity column equilibratedwith buffer D (20 mM Tris/HCl, pH 8.0) at a flow rate of 1 ml/min. Thecolumn was washed with 1.4 ml of buffer D and with 8.5 ml of bufferD/0.15 M NaCl. The XT activity was eluted with 4.2 ml of 12 mM HClfollowed by 1.2 ml of 12 mM HCl/1.5 M NaCl. Alternatively the elutionwas performed using 100 μl of antigen at 1 mg/ml in buffer D. Fractions(1 ml) were collected into tubes containing 1 ml of 0.1 M Tris/HCl, pH8.0. The XT activities of the fractions were determined.

EXAMPLE 14

Aprotinin affinity chromatography. 200 μl of desalted XT solution fromthe heparin purification step was applied at 10 ml/min to the aprotinincolumn previously equilibrated with buffer A. After washing the columnwith 6.6 ml of buffer A the adsorbed protein was eluted stepwise usingthe same buffer containing 0.3 M NaCl (10.0 ml), 0.54 M NaCl (10.0 ml),1.00 M NaCl (10.0 ml) and 1.50 M NaCl (2.4 ml). Example 15: Western BlotAnalysis. For western blot analysis, proteins were transferred topolyvinylidene difluoride membrane in a semi-dry instrument (Novex).After transfer nonspecific antibody binding sites were blocked with 2%BSA in 0.1 M Tris/HCl, pH 7.2, for 1 h at RT. The membrane was incubatedwith antiserum in 50 mM phosphate/0.15 M NaCl, 0.5 ml/l Tween 20, pH 7.4at 1:1000 dilution for 1 h. Bound antibody was detected using a secondanti-rabbit goat immunoglobulin coupled to horse-radish peroxidase at a1:1000 dilution. The blot was developed using 4chloro-1-naphthol.

EXAMPLE 16

Gel filtration chromatography. A sample of 100 μl from the heparinpurification step was applied at 1.0 ml/min to a TSK G3000 SW column (30cm×7.5 mm, 10 μm particle size) which had previously been equilibratedwith buffer A/0.15 M NaCl. Proteins were eluted with the same buffer.Fractions of 200 μl were collected and tested for XT activity. Columncalibration was performed using thyroglobulin (669.000), ferritin(440.000), aldolase (158.000), albumin (67.000), ovalbumin (43.000).

EXAMPLE 17

N-glycosidase F digestion. Aliquots (1 μg) of XT were digested with3,1×10⁻³ units of N-glycosidase F at 37° C. for 1 h and 12 h (Table II).The samples were then subjected to SDS-PAGE, and protein bands weredetected by silver staining.

EXAMPLE 18

Measurement of protein concentration. Protein concentration wasestimated by absorbance at 280 nm assuming E^(1%) _(1 cm)=10.0 or withthe Bicinchoninic Acid Protein Assay using bovine serum albumin as astandard.

EXAMPLE 19

PCR-based cloning of human xylosyltransferase. Degenerateoligonucleotide primers with deoxyinosine substitution were designedbased upon the amino acid sequence of peptides obtained after digestionof the isolated human XT with trypsin or Lys-C. The first strand of cDNAwas synthesized by the reverse transcription reaction using poly(A)⁺ RNAisolated from the chondrosarcoma cell line SW1353 as template andoligo(dT) as primer. The reverse transcription reaction was performed at37° C. for 2 h using 50 pmol of oligo(di) primers, 1 82 g of poly(A)⁺RNA, a 0.5 mM concentration of each DNTP, 1× RT buffer and 200 units ofRNase H deficient Moloney murine leukemia virus reverse transcriptase(Life Technologies, Eggenstein, Germany) in a final volume of 20 μl. ForPCR amplification the reaction mixture contained 4 μl of the reversetranscription reaction solution, 50 pmol of each primer, a 0.25 mMconcentration of each DNTP, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mMMgCl₂ and 2.5 units of hot start Taq polymerase (Life Technologies) in afinal volume of 50 μl. Amplification with degenerate oligonucleotideprimers was carried out by 40 cycles at 94° C. for 1 min, 48° C. for 1min, and 72° C. for 2 min, followed by a final extension step at 72° C.for 15 min. After agarose electrophoresis of the PCR products, DNAfragments were excised, subcloned into the pCR2.1 vector (Invitrogen.Groningen, Netherlands) and sequenced by the dideoxy chain terminationmethod using Taq DNA polymerase (Big-dye terminator cycle sequencingkit, Perkin-Elmer, Norwalk, Conn., USA) with an automated DNA sequencer(Applied Biosystems, Weiterstadt, Germany). Several clones weresequenced to compensate for misreading by Taq polymerase.

EXAMPLE 20

Rapid amplification of 5′ and 3 ′ cDNA ends (RACE). For amplification ofthe 5′ and 3′ ends of the XT-I and XT-II isoform cDNA RACE experimentswere performed using commercially available systems (Clontech,Heidelberg, Germany; Life Technologies) according to the manufacturers'instructions. For 3′ -RACE 1 μg placenta poly(A)⁺ mRNA (Clontech) wasreverse transcribed with a 3′-CDS primer (Clontech). PCR amplificationof the 3′ cDNA end of human XT-I was accomplished according to atouch-down PCR protocol (5 cycles: 94° C. for 30 s, 72° C. for 3 min, 5cycles: 94° C. for 30 s, 70° C. for 45 s, 72° C. for 3 min, 25 cycles:94° C. for 30 s, 65° C. for 45 s, 72° C. for 3 min) with thegene-specific primer GSP1b 5′-GTGGGTATGCAGAAGTGGGGGAAGGGAC-3′ and theUPM primer mix (Clontech). An aliquot of the first PCR was subjected tosemi-nested PCR (30 cycles) using the primer Con21115′-CCCTCCGCAATGCCTACA-3′ and the UPM primer mix. The DNA fragmentsobtained were subcloned into the vector pCR2.1 (Invitrogen) andsequenced. PCR amplification of the 3′ cDNA end of the XT-II isoform wascarried out with the primers AB10267 5′-ACTGAGGTCACGCAATACAA-3′ and UPMIusing the touch-down PCR protocol. For 5′-RACE 1 μg of placenta poly(A)⁺mRNA was subjected to a reverse transcription and 5′ tailing reactionwith the 5′-CDS primer and the SMART oligo (Clontech) according to themanufacturer's protocol. The 5′ end of the XT-I cDNA was amplified usingthe primer GSP_(—)776L 5′-GCCGCACTCAGGTGATGAAGAAGT-3′ and UPM with atouch-down PCR protocol. An aliquot was used as template in a secondsemi-nested PCR reaction with the primers GSP_(—)503L5′-ACCA-CCAGGACAAAGGCGATTCTGA-3′ and UPM. The 5′-cDNA end of XT-II wasobtained by 5′-RACE amplification using the primers ABI0315L5′-AGTCGAACAGTCCAGG-GCCC-3′ and UPM mix. A new primer for a 5′-RACEreaction was designed based upon the nucleotide sequence of the largestfragment obtained (2 kbp), and the experiment was repeated with theprimer AB5846L 5′-CACGATCTCGCACTTGGGGG-3′ as described above. Thenucleotide sequences of human XT-I and XT-II cDNA have been submitted tothe GenBank/EBI Data Bank with the accession numbers AJ277441 andAJ277442.

EXAMPLE 21

Isolation of XT-I cDNA from human brain and chondrocyte cDNA libraries.Plasmid-DNA was isolated from a human whole brain cDNA library (LifeTechnologies) and a human chondrocyte cDNA library (Clontech) and usedas template in a PCR-based approach for isolation of the 5′ end of theXT-I cDNA. Briefly, GSP776L and 5′ADLD5′-CTATTCGATGATGAAGATACCCCACCAAACCC-3′ primers were used in a PCRreaction with 1 μg of plasmid DNA as template. The DNA fragments weresubjected to agarose gel electrophoresis and 0.7-3 kb long fragmentswere excised from the gel and used as a template in a semi-nested PCRreaction with GSP_(—)503L and 5′-ADLD primers. DNA fragments were thensubcloned into the vector pCR2.1 and sequenced.

EXAMPLE 22

Cloning of rat XT-I and XT-II cDNA. A RT-PCR based approach usingprimers based upon the nucleotide sequence of human XT-I and XT-II wasemployed for amplification of XT-I and XT-II cDNA from rat. The firststrand of cDNA was synthesized by the reverse transcription reactionusing poly(A)⁺ RNA isolated from the rat liver cell line BRL3A astemplate and oligo(dT) as primer. DNA fragments obtained after PCRamplification using moderate stringent conditions were subcloned intothe vector pCR2.1 and sequenced. The 5′ and 3′ ends of the XT-I andXT-II cDNA from rat tissue were amplified using the RACE strategy withgene-specific primers as described above. The nucleotide sequences ofrat XT-I and XT-II cDNA have been submitted to the GenBank/EBI Data Bankwith the accession numbers AJ295748 and AJ295749.

EXAMPLE 23

Expression levels of the XT-I and the XT-II isoform in human tissues. AHuman Multiple Tissue cDNA Panel (Clontech) was used for the analysis ofexpression levels. Levels of amplification of the housekeeping geneglyceraldehyde-3-phosphate dehydrogenase, whose transcript is alwayspresent in the tissues at an almost constant level, were determined inparallel for quality control. For amplification of the XT-I encodingtranscript the primers 128U and 601L were used, whereas primers AB10267Uand AB12394L 5′-GGAAGAGCTGGGTGTGGAAT-3′ were employed for the XT-II. PCRreactions were carried out by 22-36 cycles at 94° C. for 30 s, 60-65° C.for 45 s and 72° C. for 2 min. Amplification of a transcript wasperformed using a serial number of cycles to find the conditions forsemiquantitative amplification, and aliquots were analyzed by agarosegel electrophoresis.

EXAMPLE 24

Transfection and transient expression of XT. For construction of aeukaryotic expression vector a DNA fragment including the known codingsequence of XT-I cDNA was amplified by PCR using XT_Exp1L5′-TITCCCGTTGAGATCCTGCT-3′ and XT_Exp3U 5′-ACAGACAGCAACAACGAGAA-3′ asprimers and placenta first-strand cDNA (Clontech) as template. The 2450bp fragment obtained was cloned into the vector pcDNA4/HisMax-TOPO(Invitrogen) resulting in the fusion of XT-I to the Xpress epitope. Theplasmid pCG227-XT-I was then transiently transfected into CHO-K1 cells.The coding region of the XT-II cDNA was amplified by PCR using theprimers AB_Exp9U 5′-AAAGGAAGGCAGAGGAAGC-3′ and AB_Exp3L5′-ACCCCTCCACTGT-CTGTAAG-3′ and placenta first-strand cDNA as template.The obtained 2440 bp DNA fragment was cloned into the expression vectorpcDNA4/HisMax-TOPO resulting in the fusion of XT-II to the aminoterminalXpress epitope. The plasmid termed pCG226-XT-II was transientlytransfected into CHO-K1 cells. 2×10⁵ cells precultured for 1 day in a 35mm diameter cell culture dish were transfected with 2 μg of plasmid DNAand 6 μl of Fugene 6 transfection reagent (Roche, Mannheim, Germany).For determination of transfection efficiency CHO-K1 cells weretransfected with 2 μg of the control plasmid pcDNA4/HisMax-TOPO-lacZ. 48h after transfection the cell culture medium was harvested. Protein Gagarose beads (Sigma, Deisenhofen, Germany) and mouse anti-Xpressmonoclonal antibody (Invitrogen) were added to the cell culturesupernatant and incubated at 4° C. for 1 h. After centrifugation at10.000 g for 1 min the absorbed proteins were twice washed with PBS andresuspended in a final volume of 50 μl. XT activity was then assayed inthe samples.

EXAMPLE 25

Characterization of the reaction products. For characterization of thereaction product of recombinant XT-I the peptide QEEEGSGGGQK, which ishomologous to the amino terminus of biknn (Brinkmann et aL, 1997, l.c.),was used as acceptor in the XT activity assay. After incubation for 75min at 37° C. the enzyme was heat-inactivated by incubation for 15 minat 65° C. and the reaction mixture was used for α- and β-xylosidasetreatment and alkaline β-elimination. For the linkage-specific digestionof the bound [¹⁴C]-D-xylose 4 mU of α-xylosidase (Seikagaku Corporation,Tokyo, Japan) or 4 mU of β-xylosidase (Sigma, Dreieich, Germany) wereadded to the samples and incubated for 60 min at 37° C. The reactionmixtures were then placed on Immobilon-AV membrane discs and allowed todry. The discs were washed with trichloroacetic acid as described aboveand the remaining incorporated radioactivity was determined againstappropriate controls. The alkaline cleavage of the O-glycosidic linkagebetween the [¹⁴C]-D-xylose and the β-hydroxyaniino acid serine wasperformed as described elsewhere (Montreuil et al., 1994). Briefly, thereaction mixture was adjusted to pH 10 with diluted NaOH and an equalvolume of cold, freshly prepared sodiumboro-hydride solution (2 Msodiumborohydride in 0.1 M NaOH) and was added. After incubation at 45°C. for 16 h the cooled solution was neutralized by adding 50% aceticacid and placed on Immobilon-AV membrane discs. After drying the discswere washed with trichloroacetic acid and the remaining radioactivitywas measured by liquid scintillation counting.

1. An isoform of UDP-D-xylose:proteoglycan core proteinβ-D-xlosyltransferase(XT).
 2. A protein comprising an amino acidsequence of the isoform of claim 1 or a portion thereof, having thebiological activity of XT.
 3. A protein of claim 1 or 2 deriving fromhuman or rat sources (hXT, rXT)
 4. An isolated protein according toclaim 3, wherein said hXT has a molecular weight of 120.000 under SDSpolyacrlylamide gel electrophoresis conditions.
 5. A recombinant proteinof claim 3, wherein said protein is hXT-I comprising at least 827 aminoacids.
 6. A protein according claim 5, having the amino acid sequence asdepicted in FIG. 7B.
 7. A protein according to claim 3, wherein saidprotein is hXT-II comprising 865 amino acids and is an isoform of hXT-I.8. A protein according to claim 7 exhibiting approximately 55% overallsequence identity to hXT-I
 9. A protein according claim 8, having theamino acid sequence as depicted in FIG. 8B.
 10. A DNA sequence codingfor a protein of any of the claims 1-9.
 11. A DNA sequence according toclaim 10 comprising the nucleotide sequence coding for hXT-I as depictedin FIG. 7A, or rXT-I as depicted in FIG.
 9. 12. A DNA sequence accordingto claim 10 comprising the nucleotide sequence coding for hXT-II asdepicted in FIG. 8A or rXT-II as depicted in FIG.
 10. 13. An expressionvector comprising a promotor sequence, a DNA sequence of claim 10 andoptionally a signal sequence.
 14. An expression host cell comprising avector of claim 13, said host cell being capable of expressing a proteinof any of the claims 1-9.
 15. An antibody directed against a protein asdefined in any of the claims 1-9.
 16. A process for isolating andpurifying a protein as defined in claims 1-4, characterized by thefollowing steps: (i) culturing cells having an increased level of XT,and harvesting the supernatant of said cell culture, (ii) fractionatedammonium sulfate precipitation of the supernatant of step (i), (iii)heparin affinity chromatography of the precipitate of step (ii), (iv)ion exchange chromatography of the step (iii) product, (v) affinitychromatography of the step (iv) product, and (vi) SDS-Polyacrylamide gelelektrophoresis of step (v).
 17. A pharmaceutical composition comprisinga protein of any of the claims 1-9 and a pharmacologically acceptablecarrier, diluent or excipient.
 18. Use of a protein of any of the claims1-9 for the manufacture of a medicament for the treatment of scleroticdiseases and chronic inflammatory joint diseases.
 19. Use of claim 18,wherein said medicament is an inhibitor or antagonist of said protein.20. Use of a protein of any of the claims 1-9 as diagnostic marker. 21.Use of a DNA molecule of any of the claims 10-12 as gene marker.
 22. Useof an antibody as defined in claim 15 in an immunological assay fordetermination of a protein having the biological activity of hXT asdiagnostic tool.